Glioblastoma multiforme (GBM) is one of the most lethal and difficult tumors to treat; intensive combinations of radio- and chemotherapy are no curative and yield only a modest impact on patient survival. There is a major need for new alternative therapeutic modalities. Local treatments such as surgical resection and stereotactic radiosurgery, have been partially successful, whereas systemic therapy has been ineffective. Previous studies by our laboratories have shown that the novel interleukin MDA-7 / IL-24 administered either as a virus (Ad.mda-7) or as a purified fusion protein (GST-MDA-7), suppressed the growth of GBM cells. Low concentrations of MDA-7 (approximately 0.5 nM) suppressed growth without killing cells whereas higher levels of MDA-7 (> 20 nM) suppressed growth and enhanced cell death. In addition, low levels of MDA-7 enhanced the in vitro radiosensitivity of established and primary GBM cells cultured in 2- and 3-dimensions, and when grown as tumors in vivo. These anti-proliferative and cytotoxic effects were not observed in primary astrocytes. The molecular mechanisms by which MDA-7 inhibits GBM cell proliferation and interacts with radiation to kill GBM cells are unknown. For the studies in Project 2, we hypothesize that MDA-7 acting as a cytokine both promotes cell death and, in addition, modulates mitochondria! function so that tumor cells are more radio-responsive. Specific aim 1 will determine the concentration-dependent effect of MDA-7 on the activation of the ERK1/2, ERK5, PI3K/AKT, JNK and P38 pathways; pathways that control the proliferation and survival of established GBM cells (U251) and non-established GBM cells (GBM6 expressing EGFR VIII, GBM14 lacking PTEN function. Parallel studies will determine the impact of radiation exposure on MDA-7-induced pathway activation. The mechahism(s) by which MDA-7 enhances radiosensitivity; potentially by altering reactive oxygen species generation and the expression of BCL-2 / BH3 family members, will be determined. Specific aim 2 will determine the molecular mechanisms by which combined treatment of GBM cells with PIS kinase and MEK1/2/5 inhibitors enhance cell killing by MDA-7. Specific aim 3 will determine whether infusion of Ad.mda-7 or infusion of GST-MDA-7, into a pre-existing primary human GBM6 tumor, reduces tumor growth and enhances radiosensitivity in vivo.